While considerable progress has been made in the prevention of heart diseases that are caused by environmental factors, such as nicotine, hypercholesterolemia or diabetes, and in the symptomatic treatment of heart conditions, there is still a need for methods that improve the treatment of inherited cardiomyopathies. Among the cardiomyopathies that are caused by genetic factors are hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC).
HCM is the most prevalent myocardial disease characterized by unexplained left ventricular hypertrophy in the absence of another cardiac or systemic disease that itself would be capable of producing the magnitude of hypertrophy evident in a given patient. HCM is associated with initially normal systolic, but impaired diastolic function (Elliott et al., 2008, Eur Heart J 29:270-276; Gersch et al., 2011, J Thorac Cardiovasc Surg 142:e153-203). HCM has a particularly high prevalence of about 1:500 in the general population (Maron et al., 1995, Circulation 92:785-789), and it is the leading cause of sudden cardiac death in younger people, particularly in athletes. Although HCM is a life-threatening disease, no curative treatment exists to date (Carrier et al., 2010, Cardiovasc Res 85:330-338; Schlossarek et al., 2011, J Mol Cell Cardiol 50:613-20).
HCM is an autosomal-dominant disease which is known to be caused by more than 1000 different mutations in at least 10 genes that encode components of the cardiac sarcomere, such as cardiac myosin binding protein C (MYBPC3), β-myosin heavy chain (MYH7), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin ventricular essential light chain 1 (MYL3), myosin ventricular regulatory light chain 2 (MYL2), cardiac α actin (ACTC), α-tropomyosin (TPM1), titin (TTN), four-and-a-half LIM protein 1 (FHL1) (Richard et al., 2003, Circulation 107:2227-2232; Schlossarek et al., 2011, J Mol Cell Cardiol 50:613-20; Friedrich et al., 2012, Hum Mol Genet 21:3237-54). Most mutations are missense mutations which encode full-length mutant polypeptides. The most known exceptions are MYBPC3 and FHL1, which exhibit mainly frameshift mutations leading to C-terminal truncated proteins.
The most frequently mutated gene in HCM is MYBPC3 which encodes cardiac myosin binding protein C (cMyBP-C) (Bonne et al., 1995, Nature Genet 11:438-440; Watkins et al., N Engl J Med., 2011, 364:1643-56). cMyBP-C is a major component of the A-band of the sarcomere, where it interacts with myosin, actin and titin (Schlossarek et al., 2011, J Mol Cell Cardiol 50:613-20). In humans and mice cMyBP-C is exclusively detected in the heart (Fougerousse et al, 1998, Circ Res 82:130-133) and is involved in the regulation of cardiac contraction and relaxation (Pohlmann et al., 2007, Circ Res Circ Res 101, 928-38; Schlossarek et al., 2011, J Mol Cell Cardiol 50:613-20). About 70% of the mutations in the MYBPC3 gene result in a frameshift and produce C-terminal truncated proteins (Carrier et al., 1997, Circ Res 80:427-434). Truncated proteins are unstable and have never been detected in myocardial tissue of patients (Marston et al., 2009, Circ Res 105:219-222; van Dijk et al., 2009, Circulation 119:1473-1483; van Dijk et al., 2012, Circ Heart Fail 5:36-46).
Therefore, a reduced level of cMyBP-C protein is one argument that haploinsufficiency is a likely disease mechanism of HCM. An insufficient amount of full-length cMyBP-C could produce an imbalance in the stoichiometry of the thick filament components and alter sarcomeric structure and function. Haploinsufficiency is also involved in mouse and cat models of HCM that carry either missense or frameshift mutations (Meurs et al., 2005, Hum Mol Genet 14:3587-3593; Vignier et al., 2009, Circ Res 105:239-248). In addition, in both cats and mice, there is evidence for the presence of mutant cMyBP-C (full-length or truncated), even at low level. Therefore, a second likely disease mechanism is the generation of toxic polypeptide inducing a dominant-negative effect, most probably by competing with the wild-type (WT) gene product.
Current drug-based treatments of HCM are merely empiric, can alleviate the symptoms but do not treat the genetic cause underlying the disease. Clearly, a gene-based or RNA-based therapy would be the only curative treatment for HCM. Gene therapeutic approaches have successfully been tested in connection with non-genetic cardiac diseases (Jessup et al., 2011, Circulation 124:304-313).
US applications 2005/0276804 and 2007/0292438 disclose that cMyBP-C is associated with genetic cardiac disorders. However, US 2005/0276804 suggests a reduction of retinol binding protein or retinoid to treat these disorders. US 2007/0292438 is limited to the disclosure of different mouse models having disruptions in various genes.
US applications 2004/0086876 and US 2002/0127548 disclose the diagnosis of mutations in the human MYBPC3 gene which are associated with HCM. Further, these applications suggest treating HCM by administration of a nucleic acid which encodes a non-mutated cMyBP-C to the patient.
Merkulov et al., 2012, Circ Heart Fail, 5:635-644 disclose the transfer of the murine Mybpc3 gene into the myocardium of cMyBP-C-deficient (cMyBP-C−/−) mice. The authors assume that the absence of cMyBP-C results in dysfunction and hypertrophy. The gene transfer improved systolic and diastolic contractile function and led to reductions in left ventricular wall thickness in the cMyBP-C-deficient (cMyBP-C−/−) mice.
Vignier et al., 2009, Circ Res 105:239-248 developed a Mybpc3-targeted knock-in (KI) mouse model carrying a G>A point mutation that results in different mutant mRNAs and proteins originating from abnormal gene transcription and splicing. It was shown that exogenous stress, such as adrenergic stress or aging, leads to a saturation and finally to an impairment of the ubiquitin-proteasome system (UPS) in the KI mice and potentially to a subsequent accumulation of the mutant cMyBP-C polypeptides.
The present inventors found that in subjects suffering from HCM due to a heteroallelic mutation acting in a dominant-negative fashion in a gene encoding a cardiac sarcomeric protein, the introduction of a gene transfer vector which provides the corresponding non-mutated gene not only restores normal levels of the sarcomeric protein, but also minimizes the deleterious effects of toxic mutant polypeptides that are otherwise generated through transcription of mutant allele(s).
A vector-induced expression of an exogenous wild-type (WT) gene under the control of a cardiomyocyte-specific promoter thus overcomes the dominant-negative effect of the mutant protein in a subject which carries a mutated MYBPC3 allele and is not toxic, because, surprisingly, expression of the normal allele via the gene therapy vector effectively reduces the expression of the endogenous mutant allele. This effect is considered to occur as a cardiac cell-autonomous phenomenon due to tight intracellular control of the homeostasis and turnover of sarcomeric proteins.